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Abstract

Dabigatran etexilate (DE) is the prodrug of dabigatran, a direct potent thrombin inhibitor, approved as an alternative to warfarin for prevention of strokes in patients with atrial fibrillation. However, as with any anticoagulant, bleeding risks are associated with its use. Though there is no specific antidote available, an antibody fragment (Fab) against dabigatran is currently in development to neutralize its activity. Inhibition of dabigatran and its metabolites was tested in vitro using a diluted thrombin time assay. The ability of the Fab alone to induce coagulation was tested in various assays in human plasma including endogenous thrombin potential (ETP, 5 pM tissue factor) and ecarin chromogenic assay (ECA). Dabigatran etexilate (DE), 30 mg/kg was given orally to rats every 8 hrs to achieve supratherapeutic levels of dabigatran and induce bleeding in a rat tail bleeding model. 45 min after the last dose increasing i.v. Fab doses were given to test for bleeding reversal. Ex vivo clotting and plasma levels of dabigatran and Fab were measured. The Fab potently binds dabigatran (Kd 2 pM). Anticoagulant activity of dabigatran and its metabolites was inhibited with an IC50 of 3.5 and 2.0 nM in human plasma. Fab had no effect on thrombin generation (ETP, 0.89-fold of control), in contrast FEIBA resulted in ∼2-fold increase in ETP. ECA was not elevated with Fab vs control. DE (30 mg/kg p.o.) was given in 8 hr intervals to achieve steady state levels in the rat, with supratherapeutic plasma levels (1500 nM or ~750 ng/mL). This resulted in ~2-fold increase in tail cut bleeding time. There was a rapid, dose-dependent decrease in blood loss after i.v. injection of Fab, which was maintained for 6 hrs after the highest dose of Fab. aPTT, TT and ECT were all reversed. Treating rats with warfarin (0.5 mg/kg p.o.) over 3 days resulted in a 2-fold elevation of blood loss. Similar doses of Fab had no effect on reversing this elevated blood loss. These data show the specificity and selectivity of the antibody fragment for dabigatran. Administration of the Fab after DE resulted in a safe and rapid reversal of blood loss, which correlated with reversal of ex vivo clotting tests. Thus this agent holds promise for reversing dabigatran-induced anticoagulation and bleeding effects.